Protamine in treatment of neuronal injuries

ABSTRACT

The present invention relates to treatment of neuronal injury. The present invention discloses a novel use of an agent and a novel method for promoting neurite out growth and/or neural regeneration in CNS injuries. A novel mechanism of promoting neurite outgrowth by increasing the interaction of chondroitin sulphate proteoglycan (CSPG) to receptor protein tyrosine phosphatase sigma (RPTPσ) is disclosed.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application includes an electronically submitted sequence listingin .txt format. The .txt file contains a sequence listing entitled“2016-04-08 0837-0403PUS1_ST25.txt” created on Apr. 8, 2016 and is 2,238bytes in size. The sequence listing contained in this .txt file is partof the specification and is hereby incorporated by reference herein inits entirety.

FIELD OF THE INVENTION

The present invention relates to treatment of neuronal injuries.Specifically, the present invention relates to novel means andmechanisms for promoting neurite outgrowth and/or neural regeneration indiseases in which chondroitin sulfate proteoglycans (CSPGs) have adverseeffects on neural regeneration or maintenance, such as injuries of thenervous system.

BACKGROUND OF THE INVENTION

Nervous system injuries affect millions of people every year. As aresult of this high incidence of neurological injuries, neuronalregeneration and repair is becoming a rapidly growing field dedicated tothe discovery of new ways to recover nerve functionality after injury.The nervous system is divided into two parts: the central nervous system(CNS), which consists of the brain and spinal cord, and the peripheralnervous system (PNS), which consists of cranial and spinal nerves alongwith their associated ganglia. A brain injury or brain damage is thedestruction or degeneration of brain cells in the brain of a livingorganism. Brain injuries can be classified along several dimensions.Primary and secondary brain injuries are ways to classify the injuryprocesses that occur in brain injury.

Post traumatic regeneration of the brain and spinal cord is a majorunsolved medical problem because the brain and spinal cord are not ableto regenerate like the peripheral nervous system. While peripheral axonsregenerate in patients after nerve injury, brain and spinal cord axonsfail to regenerate due to glial scar formation and the inhibitory actionof chondroitin sulphate proteoglycans (CSPGs) in the scar. In addition,those factors that promote peripheral nerve regeneration, for instancenerve growth factor, NGF, fail to improve regeneration in the brain andspinal cord. The central nervous system and peripheral nervous systemare very different in their reactions to drug treatment and regenerationability.

Identifying molecular mechanisms guiding neuronal development has been agreat challenge. Inhibition of chondroitin sulphate proteoglycans(CSPGs) as a mechanism to enhance neuronal growth has been ofconsiderable interest. CSPGs have been implicated in inhibitingregeneration of axons and dendrites following CNS trauma (Silver andMiller, 2004). CSPGs are also known to be part of the glial scar thatforms post-injury, acting as a barrier to prevent axon extension andregrowth. Levels of versican, neurocan, brevican and phosphacan (thoseCSPGs measured) have all been found to be upregulated after spinal cordinjury (Jones et al., 2003).

WO2004/103299 discloses a method of improving functional recoveryfollowing a central nervous system contusion injury. The disclosedinvention is directed to a method of utilizing chondroitinase(chondroitin sulfate degrading enzyme) to promote autonomic neurologicalfunctional recovery following injury in or to the spinal cord.Compositions useful in the method include acceptable formulations ofchondroitinase. The method includes administering a therapeuticallyeffective amount of glycosaminoglycan degrading enzyme. Theglycosaminoglycan degrading enzyme may be dermatan sulfate orchondroitin sulfate degrading enzymes. The functional recovery mayinclude autonomic functions, sensory functions, motor functions or thelike.

WO2005/087920 relates to recombinant and modified chondroitinase ABC I,their production and their uses. The disclosed chondroitinase ABC Ienzymes are useful for a variety of purposes, including therapeuticmethods such as promoting nerve regeneration, promoting stroke recovery,treating spinal cord injury, treating epithelial disease, treatinginfections and treating cancer.

Other approaches to CSPG inhibition have focused on the use ofmolecules/agents that inhibit the interaction of CSPGs with its receptorRPTPσ. WO2011/022462 discloses the use of soluble fragments of RPTPsthat bind CSPGs, thus acting as competitive inhibitors to prevent theCSPGs from binding RPTPs on the neuron. The neural cell can beassociated with an injury or neurodegenerative condition. WO2012/112953discloses methods for contacting a neuron with an agent that bindsRPTPσ, to thereby induce neuronal outgrowth of the neuron. The agent mayinduce clustering of RPTPσ and/or inhibit binding of CSPGs to RPTPσ.Examples of suitable agents are heparan sulfate proteoglycan, heparansulfate, heparan sulfate oligosaccharides, or heparin oligosaccharides.

For nervous system injuries there are substantial patient populationswith significant unmet needs, for which novel treatment options aredesperately required. There is currently no treatment for recoveringhuman nerve function after injury to the central nervous system.Secondary injury mechanisms have, so far, been predominantly targetedthrough the use of neuroprotective treatments. However, the compoundsand approaches, which have been tested in clinical trials thus far, havedisappointingly failed to demonstrate clear efficacy. Consequently, theuse of neuroprotective strategies, as the primary treatment option forcentral nervous system injuries remains in doubt and hence novelapproaches are required. Finding out mechanisms and means to promotenerve regeneration is important also clinically, as it is part of thepathogenesis of many diseases. In the hunt for neurostimulatory agentsthat promote nerve regeneration, well-defined models and analysismethods are required.

BRIEF DESCRIPTION OF THE INVENTION

An object of the invention is thus to provide novel means and mechanismsfor promoting neurite outgrowth and/or neural regeneration e.g. innervous system injuries.

A further object of the invention is to provide a method of promotingneurite outgrowth and/or neural regeneration.

The objects of the invention are achieved by the novel use of protamineor a peptide or a fragment thereof. Furthermore the objects are achievedby providing a novel method of promoting neuronal outgrowth bycontacting neuron with protamine, or a peptide or a fragment thereof.The preferred embodiments of the invention are disclosed in thedependent claims.

The invention is based on the surprising realization and unexpectedfinding that protamine changes the CSPG matrix from regenerationinhibiting to regeneration activating structure. Although protaminesulphate can bind heparin, and is used during cardiopulmonary bypasssurgery to neutralise anti-clotting effects, no other clinical uses ofprotamine have been identified either for use in the treatment ofneurodegenerative disease or otherwise.

Moreover, the present application surprisingly discloses that protaminepromotes neurite outgrowth and/or neural regeneration by increasing theamount of chondroitin sulphate proteoglycan, CSPG, binding to itsreceptor RPTPσ. It may have been expected that protamine would sequesterthe CSPGs from RPTPσ, through interaction of its basic residues with thenegatively charged sulphate side chains, but the present inventors foundthe reverse to be true. Through this novel mechanism, it may be statedthat protamine can promote neurite outgrowth by modulating CSPG matrixthat leads to its enhanced chondroitin sulphate epitope binding toPTPsigma. Hence, protamine and the novel mechanism of action areadvantageous and useful for the treatment of neuronal injuries.

Furthermore, it has surprisingly been found that the novel peptides ofthe present invention, which are derivable from protamine promoteneurite outgrowth effects which are comparable to the wild-typeprotamine.

An advantage of the invention is that protamine promotes CNSregeneration by converting CSPG-enriched glial scar into permissivemilieu for axon and dendrite growth in adult CNS. Additionally,protamine sulphate has already been clinically approved by the FDA andhence safety/toxicity concerns have been addressed.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following the invention will be described in greater detail bymeans of preferred embodiments with reference to the attached drawings,in which

FIG. 1. Alignment of protamine sequences from human (UniProtKB P04553)SEQ ID NO: 11, salmon (UniProtKB P69014) SEQ ID NO: 12, mouse (UniProtKBP02319) SEQ ID NO: 13, rat (UniProtKB P10118) SEQ ID NO: 14, horse(UniProtKB P15341) SEQ ID NO: 15, Killer whale (UniProtKB P24713) SEQ IDNO: 16 and sheep (UniProtKB P68038) SEQ ID NO: 17 show significantsimilarity. Amino acids are highlighted with greater than 70% identity(black) and similarity (grey).

FIG. 2. Protamine promotes neurite growth on CSPG-coated substrate inrat cortical neurons in vitro. Chondroitin sulphate proteoglycanaggrecan prevents attachment and neurite growth in embryonic ratcortical neurons. Protamine overcomes the inhibitory action of aggrecan,promoting neuronal attachment and neurite growth on the aggrecan-coatedsubstrate in vitro.

FIG. 3. Peptides derived from protamine promote neurite growth onCSPG-coated substrate in rat cortical neurons in vitro. Chondroitinsulphate proteoglycan aggrecan prevents attachment and neurite growth inembryonic rat cortical neurons (FIG. 3A). Peptide 2 (SEQ ID NO:3,)Peptide 12 (SEQ ID NO: 10) and also pegylated protamine overcome theinhibitory action of aggrecan, promoting neuronal attachment and neuritegrowth on the aggrecan-coated substrate in vitro.

Chondroitin sulphate proteoglycan aggrecan prevents attachment andneurite growth in embryonic rat hippocampal neurons (FIG. 3B). Peptide 3(SEQ ID NO:4) overcomes the inhibitory action of aggrecan, promotingneuronal attachment and neurite growth on the aggrecan-coated substratein vitro.

FIG. 4. Protamine increases the amount of aggrecan binding to RPTPσ.Mutant RPTPσ with a defective CSPG-binding site was used as a negativecontrol.

FIG. 5. Peptide 12 (denoted in the figure as short protamine) improvesfunctional recovery as assessed by vertical screen (A) and cylinder (B)behavior tests.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a use of protamine or a fragmentthereof for treating injuries of the nervous system in a subject.Specifically, the present invention relates to a use of protamine or afragment thereof as anti-CSPG therapy in diseases in which the CSPGsrestrict neuronal regeneration or maintenance. According to theinvention protamine promotes neurite outgrowth and neuronal attachmenton CSPG-coated substrate by overcoming the inhibitory action of CSPG(FIG. 2). Furthermore according to one embodiment of the inventionprotamine increases the interaction of chondroitin sulphate proteoglycan(CSPG) to the receptor protein tyrosine phosphatase sigma (RPTPσ) (FIG.4), thereby promoting the neurite outgrowth.

The present invention relates also to a protamine peptide consisting ofan amino acid sequence set forth in SEQ ID NO: 3, or its variant.Furthermore, the present invention relates to the use of a protaminepeptide consisting of an amino acid sequence according to SEQ ID NO: 3as a medicine.

The present invention relates also to the use of a protamine peptideconsisting of an amino acid sequence set forth in SEQ ID NO: 3 or itsvariant in treating neuronal injuries in a subject.

According to the present invention protamine or a peptide or fragmentthereof is used in treating neuronal disorders, which include disease,disorder, or condition directly or indirectly affecting the normalfunctioning or anatomy of a subject's nervous system. The disorder maybe a neuronal injury, which can be acute or chronic. Examples of acuteinjury are those that result from surgery, trauma, compression,contusion, transection or other physical injury, vascular pharmacologicor other insults including hemorrhagic or ischemic damage. Chronicneuronal injury may result from repetitive stress,inflammation/oxidative stress within a neural tissue caused by disease,neurodegenerative or other neurological diseases.

According to the present invention protamine or a peptide or fragmentthereof is beneficial in all diseases where the CSPG matrix isinhibitory for regeneration or maintenance of axons and dendrites. Inone embodiment of the present invention the disease is a neuronal injuryselected from a group consisting of neurodegenerative diseases,traumatic brain injury, spinal cord injury, multiple sclerosis (MS),amyotropic lateral sclerosis (ALS), Parkinson's disease, stroke,peripheral nerve injury, eye injury and skin burn. Preferably theneuronal injury is TBI or SCI.

Protamines are small, arginine-rich, nuclear proteins that replacehistones late in the haploid phase of spermatogenesis and are believedto be essential for sperm condensation and DNA stabilisation. Protaminehas been shown to be able to condense plasmid DNA efficiently fordelivery into several different types of cells. Protamine sulfate is acommonly used salt form of protamine.

Gene and protein sequences have been determined for protamines ofnumerous vertebrate species. Mice, humans and certain fish have two ormore different protamines, whereas the sperm of rabbits, for example,have only one form. The two human protamines are denoted by PRM1(UniProtKB P04553) and PRM2 (UniProtKB P04554), whilst examples ofprotamines from fish include salmine from salmon (UniProtKB P69014),clupeine from herring (UniProtKB P69010) and iridine from rainbow trout(UniProtKB P02328). FIG. 1 shows a sequence alignment of protaminesderived from different species and demonstrates the significant amountof conservation throughout the amino acid sequences.

Protamine sulphate can bind heparin and is mandatorily used duringcardiopulmonary bypass surgery to neutralise the anti-clotting effectsof heparin. It is estimated that over two million patients are exposedto the heparin-protamine interaction per year, where 1 mg of protaminesulphate (i.v.) is administered for every 100 IU of active heparin.Protamine neutralization of heparin can cause increased pulmonary arterypressures and decreased systolic and diastolic blood pressure,myocardial oxygen consumption, cardiac output, heart rate, and systemicvascular resistance. These multiple cardiovascular effects are mediatedvia complement activation, histamine release, thromboxane and nitricoxide production, and antibody formation (Carr and Silverman). Sinceprotamine is a highly cationic peptide, it can bind to heparin to form astable ion pair, which does not have anticoagulant activity. The complexof heparin-protamine is then removed and broken down by thereticuloendothelial system.

Protamine has also been used in gene transfer, protein purification andin tissue cultures as a crosslinker for viral transduction.

According to the present invention protamine may have eg. a sequence setforth by SEQ ID NO: 1 or any other similar sequence. The presentinvention relates also to the use of fragments of protamine. As usedherein “a fragment” refers to any part of protamine that is long enoughto have the desired activity of neurite outgrowth. In a preferredembodiment of the invention, protamine or a fragment thereof is native,i.e. a protein in its natural state, unaltered by heat, chemicals,enzyme action, or the exigencies of extraction.

According to the present invention “a variant” of protamine peptide maycomprise amino acid substitutions, deletions or insertions, but it stillfunctions in a substantially similar manner to the protamine peptidedefined above. The peptides of the present invention and variantsthereof may be fused to other proteins, polypeptides or peptides (N- orC-terminally), or conjugated to other substances. The peptides of thepresent invention may also be bound or conjugated to substances whichenhance their ability to pass through the blood brain barrier. Accordingto one embodiment of the present invention also peptides are encompassedwhich exhibit at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identitywith the peptide having SEQ ID NO:3. Whether any two amino acidmolecules have amino sequences that are at least, for example, 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% “identical”, can be determined usingknown computer algorithms such as the “FASTA” program, using forexample, the default parameters as in Pearson et al. (1988) PNAS USA 85:2444.

As used herein, the term “neurite growth” or “neurite outgrowth”includes the process by which axons or dendrites extend from a neuron.The outgrowth can result in a new neuritic projection or in theextension of a previously existing cellular process. Neurite outgrowthmay include linear extension of an axonal process by five cell-diametersor more. “Central nervous system (CNS) neurons” include the neurons ofthe brain, the cranial nerves and the spinal cord. The invention relatesnot only to CNS neurons but also to peripheral neurons that makeprojections (axons) in CNS, for instance dorsal root ganglion neurons.

As used herein the term “brain injury” is the destruction ordegeneration of brain cells is in the brain of a living organism. Braininjuries can result from direct impacts to the head. Such injuries arefor example traumatic brain injury and spinal cord injury. The presentinvention may also be used in treating other neuronal disorders, whichinclude disease, disorder, or condition directly or indirectly affectingthe normal functioning or anatomy of a subject's nervous system. Thedisorder may be a neuronal injury, which can be acute or chronic.Examples of acute injury are those that results from surgery, trauma,compression, contusion, transection or other physical injury, vascularpharmacologic or other insults including hemorrhagic or ischemic damage.Chronic neuronal injury may result from repetitive stress,inflammation/oxidative stress within a neural tissue caused by disease,neurodegenerative or other neurological diseases. The invention can bebeneficial in all diseases where the CSPG matrix is inhibitory forregeneration or maintenance of axons, such as TBI, SCI, multiplesclerosis (MS disease) and amyotrophic lateral sclerosis (ALS).

“Traumatic brain injury, TBI” as used herein includes the condition inwhich a traumatic blow to the head causes damage to the brain orconnecting spinal cord, with or without penetrating the skull. Itrelates more specifically to the actual mechanical damage that occurs atthe type of trauma, such as shearing, tearing and stretching of axons,neurons and blood vessels. Usually, the initial trauma can result inexpanding hematoma, subarachnoid hemorrhage, cerebral edema, raisedintracranial pressure, and cerebral hypoxia, which can, in turn, lead tosevere secondary events due to low cerebral blood flow.

“A spinal cord injury, SCI” as used herein is damage to any part of thespinal cord or nerves at the end of the spinal canal. It often causespermanent changes in strength, sensation and other body functions belowthe site of the injury. The spinal cord injury may be a completesevering of the spinal cord, a partial severing of the spinal cord, or acrushing or compression injury of the spinal cord. Spinal cord injurySCI proceeds over minutes, hours, days and even months after the initialtraumatic insult and can lead to significant expansion of the originaldamage. These secondary events are a consequence of delayed biochemical,metabolic and cellular changes, which are initiated by the primaryinjury, and includes inflammation, free radical induced cell death andglutamate excitotoxicity.

Axonal sprouting, from surviving neurons, is associated with spontaneousmotor and sensory recovery following TBI and SCI. Although the CNS has alimited capacity to regenerate, spontaneous pericontusional axonsprouting does take place approximately 1-2 weeks after trauma. However,this process typically fails due to an inhibitory axonal environmentpromoted by chondroitin sulphate proteoglycans (CSPGs). Astrocytes, atthe site of injury, produce CSPGs, beyond which the axons cannotregenerate (Silver and Miller, 2004). Inhibition of CSPG activityrepresents one potential approach to neuroregeneration, following eitherTBI or SCI. Evidence in support of this theory has been provided throughthe use of chondroitinase ABC (ChABC, an enzyme that degrades CSPGs) atthe site of trauma in rodent models of TBI and SCI. ChABC treatmentresulted in an enhanced and prolonged sprouting response with anincrease in sensory, motor and autonomic function (Harris et al., 2010,Starkey et al., 2012).

Multiple sclerosis (MS) is a chronic immune-mediated disease that ischaracterized by demyelinating and degenerative processes within thecentral nervous system. MS potentially requires symptomatic anddisease-modifying therapies. Numerous symptoms such as fatigue,spasticity, depression, bowel and bladder dysfunction, pain, andimpaired mobility are associated with the neurologic damage that resultsfrom MS. Several therapies e.g. modafinil, dalfampridine, baclofen,diazepam, gabapentin, opioids are used for symptomatic treatment ofdisability and symptoms, but these do not improve disease outcome.Intravenous corticosteroids are used in the management of MSexacerbations, but do not appear to affect the degree of improvementfrom acute exacerbations. A more definitive therapy for MS should reducerelapse rate, prolong remission, limit the onset of new MS lesions, andpostpone the development of long-term disability. There are currentlyavailable MS disease-modifying therapies, but thus far no beneficialagent has been established in primary-progressive MS.

Amyotrophic lateral sclerosis (ALS), also referred to as motor neurondisease and Lou Gehrig's disease, is the most common form of the motorneuron diseases. The disorder is characterized by rapidly progressiveweakness, muscle atrophy, twitching and spasticity, difficulty withspeaking and swallowing and a decline in breathing ability. The definingfeature of ALS is the death of both upper and lower motor neurons in themotor cortex of the brain, the brain stem, and the spinal cord. Thedisease has its onset usually in midlife and leads to death within 3-5years from diagnosis, usually due to respiratory failure. Oncediagnosed, only 10% of patients survive for longer than 10 years. In theUS, there are approximately 30,000 ALS sufferers, with 5,000 new caseseach year. Although there are currently several ongoing clinical trialsfor novel ALS treatments, there is no curative therapy for ALS andpalliative care remains the most important means of treatment.

“Chondroitin sulphate proteoglycans, CSPGs” as used herein, represent avaried class of complex extracellular matrix macromolecules. They sharea general molecular structure comprising a central core protein withheavily sulphated sugar side chains, usually glycosaminoglycans (GAGs),attached through covalent bonds. The GAG side chains are of differentlengths, which partially define the different CSPGs e.g. aggrecan(CSPG1), versican (CSPG2), neurocan (CSPG3), brevican (CSPG7) andphosphacan. Some of these aggrecans share similar N-terminal andC-terminal domains.

CSPGs play an active role in the neural development of postnatal babies,acting as guidance cues for developing growth cones. Growing axons arefound to avoid CSPG dense areas. Similarly, CSPGs found near and aroundembryonic roof plates inhibit axon elongation through the spinal cordand direct the axons in an alternative direction. CSPGs absent on roofplates were found to attract axonal elongation (Snow et al., 1990).

According to the present invention protamine promotes neurite outgrowthon CSPG coated substrate. The CSPG-coated substrate is an experimentalmodel of a CSPG matrix. It can be made for example by coating a CSPG,like aggrecan, on a tissue culture well. Anti-CSPG effects in vivo usingchondroitinase ABC were originally found using in vitro assays (Kwok J Cet al 2011). As used herein the CSPG matrix means the type ofextracellular matrix that expresses chondroitin sulphate proteoglycans.The extracellular matrix (ECM) provides a number of critical functionsin the CNS, contributing both to the overall structural organization ofthe CNS and to control of individual cells. At the cellular level, theECM affects its functions by a wide range of mechanisms, includingproviding structural support to cells, regulating the activity of secondmessenger systems, and controlling the distribution and localconcentration of growth and differentiation factors. The brainextracellular matrix has trophic effects on neuronal cells and affectneurite outgrowth.

Only recently has a receptor, protein tyrosine phosphatase sigma (RPTPσ)been identified for the CSPGs (Shen et al., 2009). It was previouslydemonstrated that disruption of the RPTPσ gene enhanced regeneration insciatic nerves, but the mechanism by which this was achieved wasunclear. An interaction was demonstrated between both neurocan andaggrecan with RPTPσ, which was dependent upon the chondroitin sulphateside chains. RPTPσ has a conserved, positively charged, region in thefirst immunoglobulin domain that is known to interact with heparinsulphate (Arisescu et al., 2002). Mutation of a cluster of four lysineresidues in this area to alanines, reduced binding of CSPG to RPTPσ tobackground levels.

A functional effect of the RPTPσ and CSPG interaction, has beendemonstrated by using dorsal root ganglion (DRG) neurons thatconstitutively express high levels of RPTPσ. Wild-type DRG neurons werecultured, in parallel with those from mice with a targeted genedisruption of RPTPσ, in the presence of a CSPG mixture. Control DRGneuron outgrowth was reduced by approximately 50% in the presence of theCSPG mixture, but had far less effect on those neurons from RPTPσ^(−/−)mice. When the DRG neurons were challenged with purified neurocan,similar results were observed.

The role of RPTPσ in inhibiting regeneration of axons and dendrites,through CSPG interaction, following injury has also been demonstratedusing appropriate in vivo models. In PTPσ^(−/−) mice, following a dorsalcolumn crush injury, axonal extension into the lesion penumbra wassignificantly improved, compared to controls (Shen et al., 2009). Theability of corticospinal tract (CST) axons to regenerate after spinalhemisection and contusion injury in PTPσ^(−/−) mice has also beenassessed. Damaged CST fibers, in PTPσ^(−/−) mice, were found toregenerate and extend for long distances after injury to the thoracicspinal cord. In contrast, no long distance axon regeneration of CSTfibers was seen after similar lesions in wild-type mice (Fry et al.,2010).

RPTPσ is also known to bind heparan sulphate proteoglycans (HSPGs),which are similar to the CSPGs, in that they contain a protein core withheavily sulphated, negatively charged, sugar side chains. AlthoughCSPGs, through RPTPσ, have been shown to have an inhibitory effect onneuronal outgrowth, HSPGs, also acting through RPTPσ, have been shown tostrongly promote neuronal growth (Coles et al., 2011). Both CSPGs andHSPGs bind to a common site on RPTPσ and these differential effects wererationalised based on RPTPσ oligomerisation status. Heparan sulphateGAGs were found to induce oligomerisation of RPTPσ fragments, butchondroitin sulphate GAGs did not support clustering. HSPGs and CSPGsdiffer in the composition of their GAG chains; sulphate groups are moreevenly distributed in CSPGs, whereas HSPGs contain areas of highsulphation. Receptor oligomerisation may cause microdomains with highphosphotyrosine levels and support neuronal extension, which CSPGs areable to disrupt and hence inhibit axon growth (Coles et al., 2011).

In the present invention the protamine or a peptide or fragment thereofis used for treating of neuronal injuries. Treating and treatment refersto increasing, enhancing and promoting neuron regeneration and/or nervegrowth in the presence of a neuronal injury. Treating and treatmentencompass both therapeutic and prophylactic treatment regimens.

According to one aspect of the present invention protamine or thepeptide or fragment thereof may be formulated in a pharmaceuticalpreparation, which can be administered to a subject for preventing ortreating neuronal injuries. The pharmaceutical preparation may furthercomprise any other therapeutically effective agents and any other agentssuch as pharmaceutically acceptable excipients, carriers, buffers,adjuvants, antiseptics, fillings, stabilizing and thickening agents andor any components well known in the art.

In a preferred embodiment of the present invention a subject is a humanor and animal.

The present invention also relates to a method of promoting neuronaloutgrowth and/or neural regeneration. This is achieved by contacting theneuron with a therapeutically effective amount of protamine or afragment thereof or the peptide of the invention. The contacting canoccur in vitro or in vivo to the neuronal cell. In vitro, protamine canbe added to a cell culture containing the neuron. In vivo protamine canbe administered to a subject, such that an effective amount of it comesin contact with the neuron to thereby induce the neuronal outgrowth. Inone embodiment, the neuron is a central nervous system neuron.

The term “administered” or “administering” to a subject includesdispensing, delivering or applying the agent to the subject by anysuitable route for delivery of the agent to a site in the body whereneuronal outgrowth is desired. Protamine can be administered directly tothe nervous system (particularly to the site of injury), intracranially,intraspinally, intracerebroventricularly, intravenously, topically orintrathecally, e.g. into a chronic lesion of a neurodegenerative diseaseor at the site(s) of traumatic injury.

The term “administered” or “administering” to a subject includesdispensing, delivering or applying the agent to the subject by anysuitable route for delivery of the agent to a site in the body whereneuronal outgrowth is desired. Protamine may be delivered according toany known method in the art. These include, without limitation,subcutaneous, intramuscular, transdermal, intravenous, oral, sublingual,nasal, rectal and topical administrations. In one embodiment of theinvention PEGylation of protamine is used to modify pharmacokinetics ofa drug and its interaction with living tissues. PEGylation of protaminesulfate enhances its ability to promote neurite growth on CSPG-substratein cortical neurons.

One preferred method of administration is to introduce protamine or apeptide or fragment thereof to the site of injury in a surgicaloperation. Another preferred method of administration is to introduceprotamine to the site of injury without open surgery, e.g. by injection.Preferably protamine is administered via direct injection orprotamine-bound carrier (scaffold) material implantation or viral vectorexpressing protamine under a cleavable secretion signal engineered inthe construct. The scaffolds may include natural components (aggrecan,neurocan, hyaluronic acid) or artificial materials. The route ofadministration and the dosage regimen will be determined by skilledclinicians, based on factors such as the exact nature of the conditionbeing treated, the severity of the condition, and the age and generalphysical condition of the patient.

In one embodiment, administration is to thereby contact injured and/ornon-injured neurons proximal to the injury site. In one embodiment,administration is such as to deliver the agent across the blood brainbarrier. The agent of the present invention may be formulated as part ofpharmaceutical compositions comprising one or more of the specificagents.

The term “effective amount” or “therapeutically effective amount” refersto the amount of an active agent sufficient to induce a desiredbiological result e.g. promotion and/or restoration of neuronalregeneration and/or neurite growth. That result may be alleviation ofthe signs, symptoms, or causes of a disease, or any other desiredalteration of a biological system.

Neuronal outgrowth induced by the methods described herein can bedetermined by a variety of methods, such as by determination of theformation of axons (e.g., detecting the formation of neuronal branchingmicroscopically or by showing cytoplasmic transport of dyes). Neuronaloutgrowth can also be detected by determination of the formation ofneural connectivity. Outgrowth can also be determined by an increase ora restoration of function of the neuron. Neuronal function can bemeasured by standard assays such as detection of action potential ornerve impulse condition by standard assays.

EXAMPLES Reagents and Chemicals

Protamine (salmon) and protamine sulphate (herring) were from Sigma,protamine sulphate (salmon) was from Leo Pharma. Bovine aggrecan wasfrom Sigma. The DNA sequence (the template cDNA clone MGC: 63375 IMAGE:6834684) of the CSPG-binding domain of mouse RPTPσ was used forFc-tagged RPTPσ protein production. Peptides derived from the protaminesequence were custom synthesised by CASLO ApS (Denmark).

Neurite Outgrowth Assay

Cortical or hippocampal neurons from E17 rat embryos were plated at50,000 cells/cm2 on plastic cell culture plates. Plates were precoatedwith aggrecan or IgG (10 μg/ml). Protamine, or derived peptides, wereprecoated together with aggrecan or added to culture medium when platingneurons. Cells were cultured for 1.5-3.5 days and after that images ofcell cultures were taken using phase contrast microscope with ×20objective. Neurite length was quantified using ImagePro software. Mouseneurons were immunostained with anti-tubulin βIII antibodies and imagedusing fluorescent microscope.

RPTPσ Binding Assay

The Fc-tagged extracellular domain of RPTPσ was immobilized on proteinG-coated plates. Binding of biotinylated aggrecan to RPTPσ wasquantified via colorimetric assay using streptavidin-conjugated horseradish peroxidise. Experiments were done by using protein G coated 96well plates (Pierce, prod. #15133). Wells were first washed briefly withPBS, 0.05% Tween-20 solution. RPTPσ wild type and mutant FC-fusionproteins were diluted into 2 μg/ml solution in buffer containing PBS, 1%BSA, 0.05% Tween-20. 150 μl of protein solution was added into the wellsand plates were left for shaking at +RT for 1 hour. Wells were washed 3times 5 minutes with 200 μl of PBS, 0.05% Tween-20. During the coatingand washing steps biotinylated Aggregan was diluted to 5 μg/ml withdifferent amounts of protamine (0-5 μg/ml) in solution containing PBS,1% BSA, 0.05% Tween-20. Aggeregan and protamine were left together withshaking for 30 minutes before applying 150 μl of this solution into thewashed RPTPσ coated wells. Wells were left for shaking at +RT for 1hour. Wells were washed 3 times 5 minutes with 200 μl of PBS, 0.05%Tween-20. Streptavidin-Peroxidase Polymer (SIGMA, S2438) was diluted1/10 000 into PBS, 1% BSA, 0.05% Tween-20 solution and 150 μl wasapplied into the washed wells which were left for shaking +RT for 30minutes. Wells were washed 3 times 5 minutes with 250 μl of PBS, 0.05%Tween-20. Detection of bound Streptavidin-Peroxidase Polymer was done byadding 200 μl of OPD (o-Phenylenediamine dihydrochloride, SIGMA P9187)substrate into the wells and was measured by reading the absorbance at450 nm (A450).

Example 1—Protamine Promotes Neurite Outgrowth on Aggrecan Substrate InVitro

Chondroitin sulphate proteoglycan aggrecan prevents attachment andneurite growth in embryonic rat cortical neurons in culture. Protamineovercame this inhibitory action of aggrecan, promoting neuronalattachment and neurite growth on the aggrecan-coated substrate in vitro.Protamine was active as both the free protein and as sulphate salt (FIG.2).

Example 2—A Peptide Derived from the Protamine Sequence Promotes NeuriteOutgrowth on Aggrecan Substrate In Vitro

Due to the activity displayed by protamine, a series of peptides (Table1), based on the protamine sequence, were synthesised and tested in theneurite outgrowth assay. Peptides 2 (SEQ ID NO:3) and peptide 12 (SEQ IDNO. 10) displayed activity (FIG. 3).

TABLE 1 Peptides derived from the protamine sequence Peptide SequenceProtamine (salmon), MPRRRRSSSRPVRRRRRPRVSRRRRRR SEQ ID NO: 1 GGRRRRPeptide 1, SEQ ID: 2 ARRRRS Peptide 2 SEQ ID: 3SRPVRRRRRPRVSRRRRRRGGRRRR Peptide 3 SEQ ID: 4 SRPVRRRRRPRVSPeptide 4 SEQ ID: 5 SRRRRRRG Peptide 5 SEQ ID: 6 RRRRRRPeptide 6 SEQ ID: 7 RRRR Peptide 7 RR Peptide 10 SEQ ID: 8 AKKKKSPeptide 11 SEQ ID: 9 KKKKKK Peptide 12 SEQ ID: 10 VSRRRRRRGGRRRR

Example 3—Protamine Increases Aggrecan Binding to RPTPσ

Aggrecan binding to RPTPσ was assessed via ELISA using Fc-tagged RPTPσand biotinylated aggrecan. Binding was quantified usingstreptavidin-bound horse radish peroxidase. Mutant RPTPσ, with adefective CSPG-binding site, was used as a negative control. Protaminewas found to increase aggrecan binding to wild type RPTPσ, by two fold,in a concentration-dependent manner. By contrast, aggrecan binding tomutant RPTPσ was not affected by protamine (FIG. 4).

Example 4—Pegylated Protamine Sulphate has Higher Activity to PromoteNeurite Growth than Protamine Sulphate

PEGylation of pharmaceutical compounds is often used to modifypharmacokinetics of a drug and its interaction with living tissues. Hereit is demonstrated that protamine keeps active upon PEGylation so thatPEGylated protamine induces neurite growth on CSPG-coated substrate(FIG. 3). The pegylation was done according to the following procedure:Protamine was buffered with 1×PBS in concentration of 9 mg/ml; 10 timesmolar excess of mPEG-NHS (Nanocs Inc.) was added on the Protaminesolution; crosslinking of mPEG-NHS to Protamine was carried out 2 hoursin room temperature with shaking; excess of mPEG-NHS was quenched byadding 100 mM Tris-HCl pH 7.5 for 1 hour in room temperature withshaking and PEGylated Protamine was purified with Heparin-sepharosechromatography by using salt gradient for elution.

Example 5—Protamine Peptide 12 Promotes Functional Recovery FollowingSCI

Nerve tracts of one side of the spinal cord were cut (hemisection) atthe C4 level, which causes reduced locomotor functions as seen invertical climbing and usage of the limb on the affected side(represented in the cylinder test). When protamine peptide 12 is givenby intracerebroventricular injection, the time used in the verticalclimbing test becomes shortened compared to injection of the vehicleonly, indicating improved basic locomotion in the group of mice treatedwith peptide 12 (FIG. 5A). Furthermore, protamine peptide 12 enhanceslimb usage on the affected side compared to the vehicle (FIG. 5B), whichalso indicates improved healing due to protamine peptide 12.

Spinal Cord Injury and Post-Operation Care

Animals were anaesthetized with a intraperitoneal mixture of Ketaminol(2 mg/20 g body weight, Intervet) and Rompun (0.25 mg/20 g body weight,Bayer). After laminectomy at C5 vertebral level, the dura was carefullyremoved. The right cervical spinal hemicord was transected with a 25Gsyringe needle from midline dorsal vessel to the lateral side. Bleedingwas controlled with haemostatic gelatin sponge. Muscle and skin weresutured with 6-0 monocryl. 30 min before surgery and during following 3days mice were treated with antibiotic Borgal (30 mg/kg, Intervet). Bodyweight and bladder expression were checked for 7 days, and animalreceived Rimadil (5 mg/kg, Pfizer) and Rapidexon (0.2 mg/kg, Evrovet) tocontrol pain and inflammatory swelling until they stop to lose bodyweight.

Behavioral Assessment of Functional Recovery

Vertical Screen (VS).

The mouse was placed on a very brink of horizontal wire screen (25×22cm, diameter of wires 2 mm spaced at 1 cm) faced to edge. Immediatelyafter that the screen was turned vertically to place mouse to upsidedown position at the lower edge. The time that animal was needed toclimb to the upper edge were measured during 1 min. This test wasrepeated every second week starting from 1st week after surgery.

Assessment of Forelimb Use.

Based on the test of abnormal posture of suspended animal applied onanimal stroke model by Hoffer (J Cereb Blood Flow Metab, 2013) andBederson (Stroke, 1986) we developed scoring system to assess theseverity of forelimb dysfunctions after cervical hemisection. Mouse washeld gently by tail suspended under the grid and then slowly pulled downto allow gripping the grid by front paws. The flexion of forelimbs insuspended position and the effectiveness and accuracy of gripping wereused for score judgment according the criteria presented in Table 2.

TABLE 2 Criteria for score judgment score criteria 0 mouse extent bothforelimbs straight in suspended position and tightly grip the grid 1mouse keep the ipsilateral to trauma site forelimb slightly flexed, butthe grid gripping is tight and accurate 2 mouse keep the ipsilateral totrauma site forelimb flexed, grid gripping is weak and mouse can makemultiple paw misplace- ment before gripping 3 mouse keep the ipsilateralto trauma site forelimb flexed and does not use it in gripping

Cylinder Test of Limb Use Asymmetry.

Mouse was placed to glass cylinder 10 cm in diameter and 14 cm high.Mirror was position at the angle behind of cylinder for betterobservation. Mouse was videotaped for the first 20 exploratory verticalrearing or for 5 min. The number of rearing in which mouse used right(impaired), left or both limbs for support against the wall weremeasured. Paw preference were calculated as ratio of total number ofleft limb use (left+both) to total number of right (impaired) limb use(right+both).

Statistic.

Data obtained from Vertical screen test were analyzed by one-way ANOVAfollowed by post-hoc Newman-Keuls test for detection of groupdifferences. Nonparametric Mann-Whitney test was used for analyses ofscore assessment of forelimb use and cylinder test.

It will be obvious to a person skilled in the art that, as thetechnology advances, the inventive concept can be implemented in variousways. The invention and its embodiments are not limited to the examplesdescribed above but may vary within the scope of the claims.

REFERENCES

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The invention claimed is:
 1. A protamine peptide consisting of an aminoacid sequence with at least 80% identity to SEQ ID NO:
 3. 2. Theprotamine peptide of claim 1, wherein the amino acid sequence has atleast 90% identity to SEQ ID NO:
 3. 3. The protamine peptide of claim 1,wherein the amino acid sequence is set forth in SEQ ID NO:
 3. 4. Amethod of promoting neurite outgrowth and/or neuronal regeneration,comprising administering an effective amount of the protamine peptide ofclaim 1 to a patient in need thereof.
 5. The method of claim 4, whereinthe subject has experienced a neuronal injury selected from the groupconsisting of neurodegenerative diseases, traumatic brain injury, spinalcord injury, multiple sclerosis (MS), amyotropic lateral sclerosis(ALS), Parkinson's disease, stroke, peripheral nerve injury, eye injuryand skin burn.
 6. The method of claim 4, wherein the administration isone or more selected from the group consisting of intracranial,intracerebrospinal, intravenous and topical.
 7. The method of claim 4,wherein the administration is one or more selected from the groupconsisting of direct injection and protamine-bound carrier materialimplantation.
 8. A method of promoting neurite outgrowth and/or neuronalregeneration, comprising administering an effective amount of theprotamine peptide of claim 1 to a neural cell in vitro.
 9. A protaminepeptide, consisting of the amino acid sequence set forth in SEQ ID NO:10.
 10. A method of promoting neurite outgrowth and/or neuronalregeneration, comprising administering an effective amount of theprotamine peptide of claim 9 to a patient in need thereof.
 11. Themethod of claim 10, wherein the subject has experienced a neuronalinjury selected from the group consisting of neurodegenerative diseases,traumatic brain injury, spinal cord injury, multiple sclerosis (MS),amyotropic lateral sclerosis (ALS), Parkinson's disease, stroke,peripheral nerve injury, eye injury and skin burn.
 12. The method ofclaim 10, wherein the administration is one or more selected from thegroup consisting of intracranial, intracerebrospinal, intravenous andtopical.
 13. The method of claim 10, wherein the administration is oneor more selected from the group consisting of direct injection andprotamine-bound carrier material implantation.
 14. A method of promotingneurite outgrowth and/or neuronal regeneration, comprising administeringan effective amount of the protamine peptide of claim 9 to a neural cellin vitro.